Pneumococcal carriage in unvaccinated children at the time of vaccine implementation into the national immunization program in Poland

We investigated pneumococcal carriage among unvaccinated children under five years of age at a time when the conjugate polysaccharide vaccine (PCV) was introduced in Poland into the national immunization program (NIP). Paired nasopharyngeal swab (NPS) and saliva samples collected between 2016 and 2020 from n = 394 children were tested with conventional culture and using qPCR. The carriage rate detected by culture was 25.4% (97 of 394), by qPCR 39.1% (155 of 394), and 40.1% (158 of 394) overall. The risk of carriage was significantly elevated among day care center attendees, and during autumn/winter months. Among isolates cultured, the most common serotypes were: 23A, 6B, 15BC, 10A, 11A. The coverage of PCV10 and PCV13 was 23.2% (23 of 99) and 26.3% (26 of 99), respectively. Application of qPCR lead to detection of 168 serotype carriage events, with serogroups 15, 6, 9 and serotype 23A most commonly detected. Although the highest number of carriers was identified by testing NPS with qPCR, saliva significantly contributed to the overall number of detected carriers. Co-carriage of multiple serotypes was detected in 25.3% (40 of 158) of carriers. The results of this study represent a baseline for the future surveillance of effects of pneumococcal vaccines in NIP in Poland.

Serotyping of S. pneumoniae isolates. All cultured pneumococci were first serotyped using the ImmuLex™ Pneumotest Kit (SSI Diagnostica, Denmark), supplemented with a PCR and gene sequencing as previously described [33][34][35] . Serotypes not identified by the above methods were subjected to the Neufeld Quellung test 36 .
Molecular detection of S. pneumoniae serotypes. DNA extracted from culture-enriched NPS's and saliva was tested with qPCR for the presence of serotype-specific sequences by testing 1.25 μl of sample in 12.5 μl reaction volume in duplicate, as described before 37 38 , for serotype 16F published by Azzari et al. 39 , for serotypes 11A/D, 18A/B/C/F, and 19F published by Pimenta et al. 40 , and for serotype 34 published by Sakai et al. 41 . A sample was considered positive for pneumococcus when the signal detected in serotype-specific qPCR was < 40 C T . All culture-enriched samples classified with molecular methods as positive and two hundred randomly selected culture-enriched samples classified as negative for S. pneumoniae (100 NPS's and saliva samples each) have been tested with qPCR for serotype detection. Assays generating a positive signal in samples classified as negative for pneumococcus were considered as unreliable. In line with previous reports 27,37 , qPCRs targeting serotypes 4 and 5 showed a lack of specificity when applied to both NPS's and saliva samples. The results of these two assays were excluded from analysis.
Statistics. The Fisher Exact, and McNemar's tests were used to determine statistical significance (p < 0.05).
The Chi-square test was used only once when sample size was too large for the Fisher test. In each place where the p value was given and it was calculated not with the Fisher test, the name of the statistical test used was added. Data on serotype prevalence detected using different methods was correlated using Spearman's correlation test for nonparametric data. Test parameters (predictive values, sensitivity, and specificity) were calculated using online calculator (https:// www. medca lc. org/ calc/ diagn ostic_ test. php).

Results
Altogether 405 children have been enrolled in the study. Of these, nine children were excluded from further analysis for either being too young (n = 4), too old (n = 4), or being diagnosed with a lower respiratory tract infection on enrolment day (n = 1). Two children were enrolled twice in the study and results of the first sampling were the only included. We report results from 394 children from whom paired NPS and saliva were collected at the same point of time.
Study population. The frequency of inclusions declined over the study years (Table 1) with half of the children enrolled by the 14th month (September 2017) of the 44-month-long project. The number of 12-23-monthold children (n = 147 of 394) was significantly higher compared with any other age group (p < 0.001), and the number of 24-35 months old (n = 102) was significantly higher compared to 36-47 month and 48-59 montholds (n = 71 and n = 74, respectively, p < 0.01).
Carriage of S. pneumoniae. Altogether, 158 (40.1%) of 394 children were identified as carriers of pneumococcus by any method used in the study. Ninety-seven children (25.4% of 394) were identified as carriers of S. pneumoniae using the conventional culture method (Fig. 1b). Among these 97 children, 94 were positive in NPS and six in saliva. When cultured, NPS samples were more likely to be pneumococcal-positive than saliva samples (McNemar's, p < 0.0001). All 394 saliva samples yielded a colony growth on GENT-agar and all these plates were harvested whereas 68 (17.3%) of 394 NPS cultures were negative for any bacterial growth. In the analysis of results, we treated these 68 NPS samples as negative for S. pneumoniae also by molecular method, as previously described 26,27,29,42 . When comparing accuracy of NPS cultures with cultures of saliva and using combined results of a viable S. pneumoniae recovery from either NPS or saliva as the reference, the 99.2% (95% CI 97.8-99.8) accuracy of testing of NPS's was higher than 76.9% (95% CI 71.6-80.3) accuracy of saliva testing (Table 2). Samples from 155 children (39.1% of 394) were identified as positive for pneumococcus with qPCRs (Fig. 1b). qPCR positives included all samples from which S. pneumoniae was cultured except for three NPS samples positive by culture for non-typeable (NT) pneumococci (Fig. 1a), and a single saliva sample from which serotype 24F isolate was cultured (Fig. 1c). Similar to conventional culture, with molecular methods the number of positive results was higher for NPS's compared to saliva (121 or 30.7% vs. 93 or 23.6%; McNemar's, p < 0.01) (Fig. 1b) www.nature.com/scientificreports/ (95% CI 0.90-1.97, p = 0.18) for DCC-attendees among these children, an elevated risk of carriage in households without a smoker could represent confounding bias of child's social interaction. There was no effect of age or sex on prevalence of carriage detected by a particular method or overall (Table 1).
Serotype carriage. Serotypes of isolates cultured from children and detected with qPCR in cultureenriched samples in the study are all listed in Table 3. Altogether, 99 isolates were obtained from 97 children, as S. pneumoniae isolates of two different serotypes were cultured from NPS's collected from two individuals. Ninety-three of these 99 isolates represented 26 different serotypes and the remaining six were classified as nontypeable pneumococci. The most common serotype among S. pneumoniae isolates were 23A and 6B cultured from 10 children each, followed by 15BC, 10A and 11A isolated from seven children each, and serotypes 23B and  The qPCR assays applied to detect serotype-specific sequences in DNA extracted from culture-enriched samples did cover serotypes of 79 or 84.9% out of 93 encapsulated S. pneumoniae isolates cultured in this study. Among remaining 14 isolates were S. pneumoniae of serotype 35F (n = 6), 24F, 28F, 31 (n = 2 each), 35A and 37 (single isolate each) ( Table 3) Among 158 children classified as carriers of S. pneumoniae by any method, the most common serotype/ serogroup detected with qPCRs were: serogroup 15 (n = 24 or 15.2% of 158 children), serogroup 6 (n = 22 or 13.9%), serogroup 9 and serotype 23A (n = 17 or 10.8%, each). Since the number of serotype carriage events Table 3. Serotypes of strains cultured in the study or detected using serotype-specific and serogroup-specific qPCRs. # An assay of insufficient specificity thus considered to be not reliable (NR), & serotypes not targeted by qPCR, thus detected by culture alone, @ non-typeable S. pneumoniae strains, a number of S. pneumoniae strains cultured from 97 carriers, b number of serotypes detected in 155 individuals identified as carriers of S. pneumoniae by molecular method (qPCR), c number of serotypes detected in 158 individuals identified as carriers of S. pneumoniae by any method used in the study, PCV10 serotype targeted by PCV10 and PCV13, PCV13 serotype targeted only by PCV13.   www.nature.com/scientificreports/ detected with qPCR was higher compared to the number of isolates of corresponding serotypes cultured form children (n = 112 vs. n = 78 for NPS, n = 95 vs. n = 2 for saliva, and n = 168 vs. n = 79 for overall), we tested if any individual serotype or serogroup was overrepresented in qPCR results. The only significant difference between the rate of samples positive in qPCR compared to the rate of corresponding serotype/serogroup isolates cultured was observed for serogroup 9 (Fig. 2a,c). With that in mind, there was an overall agreement between the rates of samples positive for serotype/serogroup via molecular method versus culture (Fig. 2). For NPS's, the number of samples positive for serotype or serogroup by culture were concordant with the number of samples positive for the same serotypes by qPCR (Spearman's rho = 0.855, p < 0.0001) (Fig. 2a). There was also agreement between the number of serotype carriers detected with qPCR in NPS's and in saliva (Spearman's rho = 0.667, p < 0.01) (Fig. 2b), as well as between the numbers of serotype-carriers detected with qPCR in NPS's or in saliva versus overall cultured in the study (Spearman's rho = 0.771, p < 0.0001) (Fig. 2c).
Although the absolute number of VT isolates cultured from children attending DCC was significantly higher compared with children staying home only for PCV13-VT (n = 20 isolates cultured from 207 DCC attendees vs. n = 6 isolates cultured from 187 children staying home, p < 0.05), there were no differences for PCV10-VT (n = 17 isolates cultured from 207 DCC attendees vs. n = 6 isolates cultured from 187 children staying home, p = 0.051) and in fractions of VT isolates among all n = 71 cultured from DCC attendees compared with fractions among all n = 28 cultured from children staying home (17/71 or 24.0% vs. 6/28 or 21.4% for PCV10-VTs, p = 1; and 20/71 or 28.2% vs. 6/28 or 21.4% for PCV13-VTs, p = 0.62). Although the DCC attendees were more likely to be carriers, the serotype distribution was not different between children attending DCC and staying home. None of the other demographic or environmental factors were associated with differences in serotype carriage.
Co-carriage of multiple serotypes. The presence of two or more serotypes was detected in 40 (25.3%) of 158 children identified as carriers by either culture or using piaB and lytA qPCRs (Table 4). Co-carriage of two or more serotypes/serogroups was detected in 17.7% (22 of 124) NPS's and 25.5% (20 of 94) saliva samples classified as positive for S. pneumoniae by any method. There was no significant difference in the prevalence of co-carriage in NPS's and saliva samples (p = 0.60). Also, there were no statistically significant differences in cocarriage according to age (  Temporal changes in serotype carriage. To assess the impact of the implementation of PCV into the NIP in January 2017 on the VT serotypes carriage in unvaccinated children, we tested for temporal changes in the fraction of PCV10-VTs and PCV13-VTs among cultured pneumococci. We assumed that herd effects of PCV implementation will lead to a decrease of VT carriage after the vaccine roll-out. However, there were no significant differences in the rate of PCV10-VTs and PCV13-VTs before (4/30 or 13%, for both PCVs) and after January 2017 (19/69 or 28% and 22/69, or 32% respectively). Instead, there was a trend towards an increase in the VTs fraction over time (p = 0.19 and p = 0.08 for PCV10-VTs and PCV13-VTs, respectively). Also, there were no differences in the fractions of PCV10-VTs and PCV13-VTs up to (8/50 or 16% and 9/50 or 18%, respectively) and after (15/50 or 30% and 17/50 or 34%, respectively) the 50th of 99 isolates had been cultured in the study in October 2017. The trend towards an increase in VTs fraction over time was still present (p = 0.15 and p = 0.11 for PCV10-VTs and PCV13-VTs, respectively).

Discussion
There is little data published on pneumococcal carriage in Poland. Over the past two decades reports of only two Polish studies investigating the carriage of S. pneumoniae have been published. The first study was conducted between November 2000 and May 2001 in the capital city of Warsaw 15,16 . The second study took place between November 2002 and June 2003 in Lublin 17,18 . Both these studies have been conducted before the first of the PCVs, the heptavalent vaccine, became available commercially in the country. In light of obligatory pneumococcal vaccination for children born after the 31st of December 2016, data on pneumococcal carriage is needed. Here, we investigated S. pneumoniae carriage in unvaccinated children under five years of age in the country with PCV10 and PCV13 commercially available for over five years and at the time of PCV10 implementation into the infants' immunization program. Our first goal was to establish a baseline for future studies investigating the impact of PCV10 introduction into NIP on S. pneumoniae carriage. The second goal was to assess the suitability of molecular methods and of testing oral fluids for pneumococcal carriage detection by comparing saliva testing with the gold standard method of conventional culture of NPS.
Conventional culture was the only method used to detect S. pneumoniae in previous studies conducted in Poland between 2000 and 2003 [15][16][17][18] . The carriage rate of 25.4% we detected with that method in our cohort was lower compared with 45.7-54.6% reported by Sulikowska et al. 15,16 and 33.1-44.4% by Korona-Glowniak et al. 17,18 . However, the differences in cohorts' demographic composition, methods of sampling, and seasons of sample collection, make interpretation of differences in S. pneumoniae carriage difficult.
It is documented that DCC attendance increases pneumococcal transmission [43][44][45] . In all three Polish studies, which includes this study, the risk of pneumococcal carriage was significantly higher in children attending DCCs compared to staying at home. Hence, the higher proportion of children staying home in our study (47.5%) compared with the cohort investigated by Sulikowska et al. 15 15,16 (p > 0.1). Also, children were enrolled into our study all year long whereas in the previous two, exclusively during autumn and winter, the seasons when carriage rates are higher 45 . It seems very likely that the continuous enrolment contributed further to lower carriage rates in our study. Finally, higher S. pneumoniae carriage reported by Korona-Glowniak et al. 17,18 can be attributed to oropharyngeal sample (OPS) being cultured on the top of NPS in their study. When excluding results for OPS, there was no difference in point-prevalence of 25.4% detected with NPS's culture by us and 24.4% detected by Korona-Glowniak et al. 17,18 (n = 94 of 394 vs. n = 228 of 933, Chi-square, p = 0.88).
There was, however, a reduction in proportions of PCV10-VTs (23.2% vs. 73.4%) and PCV13-VTs (26.3% vs. 80.4%) among the isolates cultured in our study, when compared to the 2002-2003 study conducted by Korona-Glowniak et al. 17,18 prior to PCVs entering the Polish market. This reduction in VTs carriage in unvaccinated children is likely to represent a herd effect of commercial vaccination with PCVs prior to and independent from PCV10 implementation into the NIP. Interestingly, instead of a decline of PCV10-VTs carriage over the study period there was, albeit not statistically significant, an increase in a fraction of VTs among all isolates cultured. Absence of decline in VT carriage implies that our study can be considered to represent the baseline for future assessment of effects of PCVs introduction into NIP on carriage.
One noticeable result was the low prevalence of serotype 19A in carriage. Serotype 19A has been reported to emerge at various sites in replacement after PCV7 implementation 8,39 and to persist in carriage in populations with infants vaccinated with PCV10 42,46 . Here, carriage of 19A was detected in only two children and exclusively with the molecular method. With estimates of 25-30% of infants being vaccinated in Poland with PCV13 outside the NIP, the low presence of this PCV13-VT could reflect PCV13 herd effects. In our study serotype 19A ranked 19th in frequency in carriage, whereas between 2016 and 2020 (time of present study) 19A was, after serotype 14, the second most common in IPD in Polish children aged 12-59 months 7 . It indicates a high IPD cases to carriers' rate, thus high invasiveness of serotype 19A strains circulating in Poland.
Importantly, low numbers of VTs cultured from unvaccinated children make future assessment of direct effects of PCV implementation into NIP on VTs carriage difficult. The solution could be increasing the power of the next study by sampling higher numbers of subjects and/or detecting carriage of serotypes with a substantially more sensitive approach. The latter can be addressed by testing multiple samples per child 17,18 and/or employing molecular detection methods. www.nature.com/scientificreports/ In our study, application of molecular method and testing saliva on the top of NPS's increased the number of carriers detected by a factor of 1.7, from ninety-four identified by gold standard of conventional culture of NPS's to an overall number of 158. In both materials, NPS and saliva, the application of molecular methods significantly increased the sensitivity of S. pneumoniae detection. This is in line with results reported by Wyllie et al. in studies applying a similar protocol to test NPS's from children and to test NPS's and saliva samples from adults, conducted between 2014 and 2016 in the Netherlands 21,42 .
NPS is the specimen recommended by WHO in pneumococcal carriage detection in children 20 and it has been reported that NPS is a more valuable material than OPS 47 . Also in our study, the culture of NPS's was far superior to culture of saliva and 39.6% of carriers (61 of 154) identified by qPCR were detected by NPS's only. However, with 21.5% (34 of 158) of carriers detected exclusively in saliva, testing oral fluids substantially increased the number of carriers detected. In line with this finding, Korona-Glowniak et al. 17,18 reported that testing OPS along NPS's significantly increased the number of carriers detected, and that there was no difference between the number of carriers detected by culturing NPS compared with OPS 17,18 . Therefore, the optimal carriage detection might be achieved by testing from each individual multiple specimens, e.g. NPS, OPS and saliva, or a combination of any two of these.
Molecular methods appeared to be superior to conventional culture in detecting co-carriage of multiple serotypes in this study (2.1% in culture vs. 26.6% in qPCR). Wyllie et al. 42 and Kandasamy et al. 48 obtained similar levels of multiple serotype carriage using molecular methods. The higher sensitivity of any minority serotype detection in multi-serotype carriage allowed for a more detailed analysis of the occurrence of serotypes. Since available qPCR assays did not cover all serotypes, and not always distinguished serotypes within a serogroup, the number of multi-serotype carriers still might be understated.
Among our study limitations was a lack of molecular assays that would detect carriage of every circulating serotype. For example, isolates of serotypes 24F, 28F, 35A, 35F, and 38 have been cultured from children, yet none of these serotypes were targeted with qPCR. Another limitation was low resolution of certain qPCRs not discriminating between serotypes within a serogroup, with 10 out of 27 qPCR assays targeting more than one serotype. A limitation was also the low sensitivity of conventional culture. It concerns both sample types, but due to very rich bacterial growth, including many non-pneumococcal α-hemolytic colonies, it was particularly difficult to culture S. pneumoniae from saliva. With large numbers of serotype-carriage events detected exclusively with qPCR, and in the light of reports on non-pneumococcal streptococci expressing the pneumococcal capsular polysaccharides 7,49 , we paid particular attention to the specificity of assays. We addressed it by testing for serotype samples negative for S. pneumoniae and excluding the results of assays that generated a positive result (serotype 4 and serotype 5 specific qPCRs). Nevertheless, we can't exclude that some of the results represent carriage of confounded non-pneumococcal bacteria detected with qPCRs. For example, when applied to oropharyngeal and saliva samples from adults, a diminished specificity of serogroup 9-specific assay has been reported 27,37 and serogroup 9 was the clear outlier when culture data was compared with qPCR results in our study (Table 3, Fig. 2a,c). However, since we did not observe positivity in this assay among samples negative for S. pneumoniae, nor was there a difference between the number of NPS's and saliva samples positive for this serogroup by qPCR, and we are not aware of any reports on the assay's poor specificity in NPS's from children, we consider results for serogroup 9 as reliable. Finally, we acknowledge that the exclusion from analysis of performance the serotypes cultured yet not targeted by qPCRs and serotypes 4 and 5 not cultured yet targeted by assays of poor specificity may result in bias that favors molecular detection.
In summary, pneumococcal carriage rate detected in Polish children was lower compared with studies conducted prior to the introduction of commercial PCVs in the country, yet we attribute it to differences in setups of studies rather than the effect of PCVs. On the other hand, the decline in prevalence of PCV10-VTs and PCV13-VTs carriage compared with the pre-PCV period suggests strong herd effects of commercial vaccination independent of NIP in Poland. According to the results obtained in our study, NPS was a more valuable material in carriage detection in children and qPCR was the more sensitive method in pneumococcal and pneumococcal serotype carriage detection. Also, information about carriage rate and serotype distribution among unvaccinated Polish children obtained during this study can be used as a baseline in future carriage projects. The knowledge concerning the methods used in pneumococcal carriage detection gained during our study can be used for further research.